The goal of this proposal is to answer the question: How does the c-myc protooncogene regulate cellular proliferation? The c-Myc protein is a transcription factor, and a number of genes have been proposed to be targets of its regulation. However, the misregulation of these genes cannot explain the diverse biological effects of c-Myc, implying that additional target genes remain to be discovered. c-Myc has been implicated in the control of cellular proliferation, differentiation, and programmed cell death, but the mechanisms by which it exerts its activity on the cellular machinery are complex and not well understood. Our fascination with c-Myc stems not only from our interest in understanding basic molecular regulatory mechanisms, but also because the deregulation of c-Myc activity is associated with a wide range of human cancers. A genetic analysis can often lead to profound insights into molecular mechanisms, and the construction of knockout mice can be especially powerful. Unfortunately, the c-myc knockout mouse, because of its early embryonic lethality, did not yield insights into either cellular or molecular phenotypes. All attempts to recover c-myc -/- cells from homozygous embryos have been frustrated by the outgrowth of cells that express one of the other Myc family members. To overcome this problem, we used gene targeting to eliminate c-myc expression in a fibroblast cell line shown not to express the other family members. These knockout cells are the first experimental system in which c-myc loss-of-function phenotypes can be thoroughly investigated. The c-myc -/- cells are viable but display profound cell cycle defects. We have initiated a systematic molecular analysis of known cell cycle regulators in the c-myc -/- cells. Our new insights to date are: 1) loss of c-myc affects progression through both the G1 and G2 phases of the cell cycle; 2) the activity of all cyclin-Cdk complexes is affected; 3) the earliest and most prominent defect is an impairment in the activation of cyclin D-Cdk4/6 complexes; 4) c-Myc affects multiple points in the cell cycle; 5) Cdk7 has been identified as a new putative c-Myc target gene; and 6) the expression of virtually all previously proposed c-Myc target genes is unchanged in c-myc -/- cells. Our objective is to build on these results to fully elucidate how c-Myc regulates transition through the cell cycle. We propose to: construct homozygous c-myc knockouts in human cells, isolate new c-Myc target genes by differential cDNA screening, define the biochemical lesions in G1 and G2 cell cycle mechanisms that result from loss of c-Myc activity, and genetically test the physiological relevance of the observed biochemical lesions.